Isolation and Activity Analysis of Cell Wall Invertase Gene Promoter (EhcwINVP) from Elsholtzia haichowensis Sun
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摘要: 以海州香薷基因组DNA为模板,通过hiTAIL-PCR和walking技术扩增得到其细胞壁转化酶基因启动子(EhcwINVP)片段,长度为1727bp。生物信息学分析结果表明,该启动子片段中含有多个对脱落酸、赤霉素、细胞分裂素等激素以及对干旱、低温、重金属铜等逆境胁迫响应相关的顺式作用元件。将通过克隆得到的EhcwINVP序列替换pCAMBIA1301载体上驱动GUS报告基因表达的CaMV35S启动子序列,构建EhcwINVP融合GUS的植物表达载体EhcwINVP::GUS。转基因拟南芥植株的组织化学分析结果表明,海州香薷细胞壁转化酶基因启动子序列具有驱动GUS基因表达的功能,且在10μmol/L铜胁迫下,转基因拟南芥植株叶和根中的GUS活性分别约是对照组的1.7倍和1.5倍。Abstract: Cell wall invertase gene promoter, EhcwINVP (1727 bp), was amplified from the genomic DNA of Elsholtzia haichowensis Sun by hiTAIL-PCR and genome walking PCR. Bioinformatics analysis showed that the promoter fragment contained multiple cis-acting elements involved in the regulation of hormones including abscisic acid, gibberellin, cytokinin, and tolerance to drought, low temperature, and heavy metals such as copper. The pCAMBIA1301 binary vector was digested with restriction enzymes to remove the 35S promoter that drove GUS expression and replaced with EhcwINVP sequences to generate the construct EhNcwINVP::GUS. Histochemical analyses of transgenic Arabidopsis thaliana L. plants indicated that the EhcwINVP promoter was capable of driving GUS expression, and GUS activity in the leaves and roots of transgenic A. thaliana plants increased by about 1.7 and 1.5 times that of the control under copper stress, respectively.
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Keywords:
- Elsholtzia haichowensis Sun /
- Cell wall invertase /
- Promoter /
- Cis-acting elements
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